ProjectSummary ThefunctionofcytochromesP450canbeclassifiedintotwomajordivisions,thosethatoperateinxenobiotic degradationandthosethatplaycriticalrolesinsteroidhormonesynthesis.Duetothecriticalroleof cytochromeP450inhumanhealth,theseenzymesystemshaveoccupiedNIHsupportedinvestigatorsfor manydecades.Thegenerallongtermgoalsoftheresearchprogramaretogainamolecularlevelinsightinto theimpressiverangeoffunctionaldiversitydisplayedbytheseenzymes.Theessentialfocusofthework proposeddealswithtwoimportanthumanenzymesinvolvedinsteroidbiosynthesis,CYP17andCYP51,both currentdrugtargetsforvariousdiseases,includingatherosclerosisandprostatecancer,progressinthis endeavorultimatelydependsongainingadeeperunderstandingoftheirstructureandprecisereaction pathways.Inordertofullyunderstandthepertinentrelationshipofstructuretofunction,itisimportantto acquirestructuralinformationfornotonlythestableterminalstatesinareaction,butalsoforthereaction intermediates,investigationofthelatterhavingbeenhistoricallyimpeded,owingtotheirfleetingexistence. Also,thefactthatthesearemembrane?boundproteinshascomplicatedtheirinvestigationowingtoproblems associatedwithaggregation.Inordertoaddresstheseobstacles,thisresearchprogramemploysanapproach usinginnovativenanodisctechnologywhichnotonlyprovidesanenvironmentmimickingthenaturalcell membranefortheisolatedCYPenzyme,butalsoenablesthesynthesisofuniquefunctionaldyadsoftheCYP withitsnaturaloralternativereductases.Alsoofimmenseimportance,apowerfulcombinationofresonance Ramanspectroscopywiththecryoradiolysistechniquepermitstrappinganddetailedstructural characterizationofotherwiseelusive,butcrucialreactionintermediates,therebyprovidingaheretofore unattainablelevelofinsightintothestructureandfunctionoftheseenzymes.